Terpene Profiles Composition and Micromorphological Analysis on Two Wild Populations of Helichrysum spp. from the Tuscan Archipelago (Central Italy)

Two wild populations of Helichrysum (Mill.) located at Elba Island (Tuscan Archipelago, Central Italy) were morphologically and phytochemically analyzed to taxonomically identify H. litoreum (population A) and H. italicum subsp. italicum (population B). Micromorphological and histochemical analyses were performed on the indumentum using Scanning Electron Microscope (SEM) and Light Microscope (LM). Morphometric analyses on vegetative and reproductive traits were also conducted. Finally, a chemotaxonomic analysis was carried out on the terpene profile of flowers, leaves and bark tissues using gas chromatography (GC-MS). Results suggested that morphological discriminant traits were mainly in leaves and cypselae glandular tissues. Phytochemical analysis indicated that a high relative content of α-pinene and β-caryophyllene were the main markers for population A, while a high relative content of neryl-acetate, α-curcumene, isoitalicene and italicene, especially in the terpene profile of bark tissue, were the main compounds for discriminating population B. The analysis suggested that the wild population A could be mainly ascribed to H. litoreum, whilst population B is defined by H. italicum.


Introduction
The genus Helichrysum Mill. belongs to the Asteraceae family and includes over six hundred species and subspecies globally widespread [1,2]. In the Mediterranean area, there are currently about 25 species [3] some of which have very similar genetic traits despite the fact that they may show different morphological characteristics.
These conditions make critical the correct taxonomic identification of taxa such as Helichrysum italicum subsp. italicum (Roth) G. Don and Helichrysum litoreum Guss. (≡ H. angustifolium (Lam.) DC.), which are very closely taxonomically related [4,5] H. italicum subsp. italicum and H. litoreum are perennial, xerophilous and suffruticose plants [4] growing over calcareous and siliceous substrates in arid, stony, shallow soils [6]. They can show very similar morphological traits, among which is a synflorescence composed of a few tens to up a hundred gold-yellow capitula, with similarities in shape and size [4]. However, a few discriminating characters can be observed. For instance, H. italicum subsp. italicum shows a glandular indumentum composed of secretory glands with a high density on leaf abaxial surfaces and flower bracts [4,7]; these structures are responsible for the
The PCA analysis ( Figure 1) indicated that the first two principal components explained about 51% of the total variation among the assessed characters (Table S1). The two populations were separated along with the first principal component (Dim1). Variables describing leaf size (ID03, ID04), cypselae size and gland density of duplex hairs on cypselae (ID08, ID09. and ID17), and capitula width (ID09) were the most decisive traits and contributed to Dim1 (0.78, 0.40, 0.57, 0.77, 0.72, and 0.55, respectively), explaining 37% of the total variation. The second component (Dim2) accounted for 14.1% of the total variation and differentiated individuals by outermost bract size (ID10 and ID11; 0.65 and 0.55 on Dim2).

Micromorphological Analysis
In both populations, non-glandular and glandular trichomes present in the vegetative and reproductive organs were assessed ( Figure S1; Figures 2 and 3). The non-glandular trichomes were multicellular, simple, uniseriate, long filamentous hairs which were slightly twisted ( Figure S1a). The glandular indumentum was composed of multicellular and biseriate trichomes belonging to two main morphotypes ( Figure S1b-g). The club-shaped trichomes were composed of 10 (−12) cells arranged in two rows of five, or six, cells each: two basal cells, two stalk cells, from six to eight secretory cells of the club-shaped head surrounded by a subcuticular chamber storing secondary metabolites of terpenoid nature ( Figure S1b-d). The duplex trichomes were composed of two oblong cells, paired along their major axis, with the cuticular layer closely adhering to the hair surface and producing muco-polysaccharidic components (Figure S1e-g); the distinction between the two cells of

Micromorphological Analysis
In both populations, non-glandular and glandular trichomes present in the vegetative and reproductive organs were assessed ( Figure S1; Figures 2-3). The non-glandular trichomes were multicellular, simple, uniseriate, long filamentous hairs which were slightly twisted (Figure S1 a). The glandular indumentum was composed of multicellular and biseriate trichomes belonging to two main morphotypes (Figures S1 b-g). The clubshaped trichomes were composed of 10 (−12) cells arranged in two rows of five, or six, cells each: two basal cells, two stalk cells, from six to eight secretory cells of the clubshaped head surrounded by a subcuticular chamber storing secondary metabolites of terpenoid nature (Figure S1b-d). The duplex trichomes were composed of two oblong cells, paired along their major axis, with the cuticular layer closely adhering to the hair surface and producing muco-polysaccharidic components (Figures S1e-g); the distinction between the two cells of the duplex trichomes was more evident in the samples from population B (Monte Capanne) than in the samples from the population A (Capo d'Enfola).   The distribution pattern of trichome morphotypes in leaves over the two Helichrysum populations analyzed through SEM indicated that the leaves of all samples were covered by long filamentous non-glandular hairs on the abaxial and adaxial surfaces (Figure 2af). These hairs formed a dense indumentum that completely obscures the epidermises, making complex the sample preparation for light microscopy investigations. These The distribution pattern of trichome morphotypes in leaves over the two Helichrysum populations analyzed through SEM indicated that the leaves of all samples were covered by long filamentous non-glandular hairs on the abaxial and adaxial surfaces (Figure 2a-f). These hairs formed a dense indumentum that completely obscures the epidermises, making complex the sample preparation for light microscopy investigations. These appendages were responsible for the grey-silver appearance of the leaves, especially on the abaxial side.
The club-shaped glandular trichomes were present on both leaf laminas (Figure 2c,f), being more abundant on the abaxial surfaces in the interveinal regions, whereas they were scarce on the adaxial surfaces.
The distribution pattern of trichome morphotypes in capitula and cypselae analyzed in both Helichrysum populations using SEM indicated that club-shaped hairs occurred on the tips of the corolla lobes, both on the primordia and the fully developed florets in both populations (Figure 3a-e, arrows). The duplex glandular trichomes were present on the ovary surfaces (Figure 3b-f) and therefore on the cypselae surface; they showed a higher density in the population B samples in comparison to the population A ones (Figure 3c-g).
The SEM micrographs at higher resolution provided differences at a cell-surface level. The epidermis of cypselae of both populations showed cells organized in simple rectangular reticulate, with regular or curvilinear margins. However, clear differences between the two populations were observed on the outer surface of the cells. Specifically, the outer surface of the cells of cypselae collected at Capo d'Enfola (A) was densely covered with epicuticular waxes in the form of small papillae ( Figure 3d). By contrast, the outer surface of the cells of cypselae collected at Monte Capanne (B) lacked epicuticular waxes ( Figure 3h).

Terpene Analysis
Terpene data for bark, flowers, and leaves tissues of the two Helichrysum populations are reported in Table S2 A total of 36 compounds were identified and included 22 monoterpenes ( Figure 4) and 14 sesquiterpenes ( Figure 5).
Plants 2022, 11, x FOR PEER REVIEW 7 of 19 appendages were responsible for the grey-silver appearance of the leaves, especially on the abaxial side. The club-shaped glandular trichomes were present on both leaf laminas (Figures 2 c, f), being more abundant on the abaxial surfaces in the interveinal regions, whereas they were scarce on the adaxial surfaces.
The distribution pattern of trichome morphotypes in capitula and cypselae analyzed in both Helichrysum populations using SEM indicated that club-shaped hairs occurred on the tips of the corolla lobes, both on the primordia and the fully developed florets in both populations (Figure 3a-e, arrows). The duplex glandular trichomes were present on the ovary surfaces (Figure 3b-f) and therefore on the cypselae surface; they showed a higher density in the population B samples in comparison to the population A ones (Figure 3cg).
The SEM micrographs at higher resolution provided differences at a cell-surface level. The epidermis of cypselae of both populations showed cells organized in simple rectangular reticulate, with regular or curvilinear margins. However, clear differences between the two populations were observed on the outer surface of the cells. Specifically, the outer surface of the cells of cypselae collected at Capo d'Enfola (A) was densely covered with epicuticular waxes in the form of small papillae ( Figure 3d). By contrast, the outer surface of the cells of cypselae collected at Monte Capanne (B) lacked epicuticular waxes ( Figure 3h).

Terpene analysis
Terpene data for bark, flowers, and leaves tissues of the two Helichrysum populations are reported in Table S2 A total of 36 compounds were identified and included 22 monoterpenes ( Figure 4) and 14 sesquiterpenes ( Figure 5).
In flower tissues, the most abundant and highly significant compounds were αylangene, β-caryophyllene, β-himachalene and δ-cadinene. The α-ylangene (9.7%), βcaryophyllene (18.2%) and δ-cadinene (13.1%) showed, on average, a higher relative amount in population A than in population B, where only a few traces of these compounds were present. The relative amount of β-himachalene was, on average, higher in population B (34.3%) than in population A (22.3%). Trans-bergamotene and β-cucumene were found only in the floral tissues.
In leaf tissues, α-copaene, β-caryophyllene, β-himachalene and α-curcumene were the most abundant and highly significant compounds. In population A, α-copaene (16.1%) and β-caryophyllene (13.5%) were higher than in population B, where these compounds were found in traces. In population B β-himachalene (29.2%) and α-curcumene (15.1%) were, on average, higher than in population A (16.3% and 5.4%, respectively). In addition, a sesquiterpene named Sesqui6_UNK was detected only in the flower tissues, and was more prevalent in population A (6.2%) than in population B, where it was found only in traces.

Multivariate Analysis of Terpene Data
In flower tissue (Figure 6a, Table S3), the two principal components explained 47.7% of the total variation among compounds. The most significant compounds were neryl-acetate, α-ylangene, δ-cadinene, β-caryophyllene and α-pinene (Dim1: 0.73, 0.67, 0.55, 0.53 and 0.48, respectively), explaining 32.5% of the total variation. The second component explained 14% of the total variation, with β-himachalene providing the major contribution (Dim2: 0.41). and β-caryophyllene (13.5%) were higher than in population B, where these compounds were found in traces. In population B β-himachalene (29.2%) and α-curcumene (15.1%) were, on average, higher than in population A (16.3% and 5.4%, respectively). In addition, a sesquiterpene named Sesqui6_UNK was detected only in the flower tissues, and was more prevalent in population A (6.2%) than in population B, where it was found only in traces.
Moreover, the second component explained 13.6% of the total variation, with p-cymene observed to provide the higher contribution (Dim2: 0.68).

Morphological Analysis
The morphological analysis suggested the possibility of distinguishing the two investigated populations, especially when cypselae and leaves were deeply assessed. In population A, a more pronounced leaf width was observed compared to population B, whilst in this latter, a more undulated leaf margin was observed. These differences in leaf characteristics suggest that population A was closer to H. litoreum, while population B was closer to H. italicum.
Other differences in morphological traits were found in the duplex-hairs density and the size of cypselae. Specifically, in this study, the cypselae in population A were found to be more elongated and with fewer duplex hairs than in population B. These observations were confirmed by the literature, where Pignatti [6] indicated that the cypselae of H. litoreum were bigger and with lower duplex hairs on the surface compared to those of H. italicum.
However, differences between populations were less clear when leaves and capitula were investigated. The literature suggested that the number of capitula per synflorescences was between 40 to 160 for H. litoreum [4], whilst in this study, the capitula per synflorescences collected in population A were between 10 and 24 (17.53 ± 6.55 mm). In addition, the length and width of leaves (21.53 ± 4.5 mm; 0.91 ± 0.2 mm) and capitula length (3.1 ± 0.24) were larger in population A compared to population B (16.82 ± 4.51 mm; 0.63 ± 0.15 mm; and 2.68 ± 0.37 mm, respectively), disagreeing with the literature [4].
The smaller size of the cypselae, the leaves and the capitula observed in population A than in population B may be due to a morphological variation caused by climatic conditions. This was suggested in the literature for H. stoechas [4] and other South African species [38]. Another hypothesis describes the presence of hybridized individuals, which may increase the complexity of the detection of specific individuals [1,4,39,40].
Another characteristic suitable for discriminating the two populations was the glands on the leaf's abaxial surface [4,5]. In H. litoreum, the glands were expected to be sparser than in H. italicum, where they usually cover 70% of the indumentum [5]. This characteristic was found also in this study, where the glands on the leaf's abaxial surface of the floral branch in population A were lower than those observed in population B.

Morphological Analysis
The samples of both populations were characterized by the external micromorphology of non-glandular and glandular trichomes, their histochemistry and distribution pattern on the investigated plant organs. The major differences referred to the density of the duplex hairs on the ovary/cypselae surfaces and the observation that the twin-celled structure was more evident in population B compared to population A. This observation was confirmed by the morphological analysis, as the density of the duplex hairs was a discriminating factor between the two populations. The duplex hair density was a discriminating factor for some species of Helichrysum [5,41] but it only partially described H. italicum and H. litoreum [4,6].
The long filamentous non-glandular trichomes were ubiquitous in all Helichrysum species examined, thus indicating morphological similarities within the genus. Based on external morphology and histochemistry, the glandular trichome morphotypes were similar to those previously described in other Helichrysum species such as H. stoechas [7], H. splendidum [30] and H. italicum [42]. Specifically, the club-shaped trichomes were found to be the main sites of production of terpene volatiles components, whereas the duplex trichomes on ovaries and cypselae were typically myxogenic structures secreting mucilaginous substances when moistened in water, favoring its absorption.
Moreover, in population A, cypselae were densely covered with epicuticular waxes in the form of small papillae, suggesting the findings of Salmeri et al. [35] for H. litoreum. By contrast, a substantial absence of small papillae on the surface of the cypselae was observed in population B. Again, Salmeri et al. [35] indicated that cypselae without epicuticular waxes can be found only for H. italicum subsp. siculum, while H. italicum subsp. italicum was not investigated. Therefore, since H. italicum subsp. siculum was found only in Sicily [5], the Helichrysum from population B can be assumed to belong to H. italicum. However, despite further literature reporting the presence of both H. italicum subsp. italicum and H. italicum subsp. microphyllum at Monte Capanne [33,43], the sole micromorphological analysis was not able to surely discriminate between these two species.
Therefore, neryl acetate and a-pinene could be used as markers for H. italicum and H. litoreum in these areas. However, these compounds showed different dynamics in other areas. For instance, in Sicily [31,51], Crete [52], and Portugal [42], high concentrations of α-pinene were observed, and low or missing neryl acetate content, whilst in Croatia, their concentration was similar [53,54]. This behavior suggested that environmental conditions such as soil characteristics and climate may affect the concentration of these compounds, making challenging the discrimination between these two species on a large scale (i.e., across the Mediterranean basin).
In leaf tissues, the concentration of α-pinene and neryl acetate of both populations was similar to that observed in floral tissues. However, despite few studies analyzing leaf tissues, our results were found to be in agreement with the current literature, which indicated higher α-pinene concentrations in H. litoreum than in H. italicum [31,44]. By contrast, the concentrations of limonene and (E)-β-ocimene found in leaf tissues of population B and almost lacking in population A disagree with current literature, where limonene was found variable and (E)-β-ocimene was low or not present in the leaves of either H. litoreum and H. italicum [31,33,44,55]. Given this contrasting agreement with the literature, these compounds cannot be considered suitable for the chemotaxonomic identification of the two species.
In bark tissues, a high concentration of α-pinene was observed in population A, and very low neryl-acetate was found in population B. In addition, population B showed a higher relative limonene content than population A. To our knowledge, there is no literature reporting the pattern of monoterpenes in bark tissues of Helichrysum; however, the chemotaxonomic approach in bark tissue was performed for the genus Picea and Abies [22,56,57], suggesting that α-pinene, limonene and other monoterpenes in this plant compartment could be used as biochemical markers to identify different taxa. Overall, α-pinene, neryl acetate and limonene could be the most suitable markers in chemotaxonomic analysis to distinguish the two analyzed Helichrysum species.
Concerning the sesquiterpenes, in flower tissues of population A, the highest concentration was found for α-ylangene, δ-cadinene, and β-caryophyllene, while in population B these compounds were found only in traces and the highest concentration was found for β-himachalene. These results only partly agreed with the literature for H. litoreum, where a high content of β-caryophyllene was found, while α-ylangene, δ-cadinene and β-himachalene were almost lacking [31,44]. However, the low relative content of β-caryophyllene, δ-cadinene, and α-ylangene observed in H. italicum agreed with several scientific studies [6,33,47,53]. These dynamics suggested that the high presence of β-caryophyllene may be considered a possible marker for detecting H. litoreum, whilst the low presence of β-caryophyllene, δ-cadinene, and α-ylangene may detect the presence of H. italicum.
In leaf tissues, a very high concentration of β-caryophyllene was found in population A, while a high percentage of α-curcumene was observed in population B. For H. litoreum, studies confirmed the presence of a significant amount of β-caryophyllene and traces of α-curcumene in leaves [31,44]. For H. italicum, Bianchini et al. [58], and Giuliani et al. [33], reported low content of β-caryophyllene in Tuscany and Corsica, whilst Maggio et al. [31] found a higher concentration of β-caryophyllene in Sicily and Sardinia. The concentration of α-curcumene in H. italicum was observed to be more stable within different environments [59], resulting as a possible marker for detecting H. italicum rather than β-caryophyllene, which may be influenced by environmental conditions.
In bark tissues, β-caryophyllene was the main marker for population A, while α-curcumene was for population B. In population B, a higher percentage of the sesquiterpenes named italicene and isoitalicene compared to the other analyzed tissues was observed. As reported for monoterpenes, to our knowledge there are no studies analyzing sesquiterpenes in bark tissues of Helichrysum, and the analysis of sesquiterpenes could be considered a suitable approach to identifying specific populations [60,61]. For instance, Costa et al. [42] and Andreani et al. [50] reported the presence of italicene and isoitalicene in floral tissues of H. italicum, whilst several studies suggested the presence of these sesquiterpenes in other subspecies [31,48,[62][63][64]. By contrast, these compounds were missing in H. litoreum [31,44]. Terpene analysis of bark tissue suggested that α-curcumene, isoitalicene, and italicene could be useful to identify H. italicum, while β-caryophyllene may be considered a possible marker of H. litoreum.

Study Area
Elba island (42 • 46 58 N; 10 • 17 11 E) is the largest island of the Archipelago Toscano National Park (Livorno, Italy). The study area chosen was the only one where distinct populations of H. italicum subsp. italicum and H. litoreum were established [36,37,43].
The island landscape is heterogenous, with mountains in the western part, and plain areas in the central part of the island. The remaining areas are coastal and hilly, these latter characterized by large iron deposits. The climate is typically the Mediterranean, with warm and windy summers and mild winters. The coldest month is January, with a monthly mean temperature of 5.5 • C, while the warmest is August (27 • C). The long-term total annual rainfall average is less than 700 mm, with rainfall events mainly concentrated in autumn [65].
The two populations of Helichrysum spp. were collected in well-distinguished locations ( Figure 7). Helichrysum litoreum was collected at Capo d'Enfola (site A), a coastal area (0-30 m a.s.l) in the north part of the island with south-west exposure. Helichrysum italicum subsp. italicum was collected in the eastern part of the island at Monte Capanne (site B), the highest peak of the island (1018 m a.s.l).

Plant Materials
A total of 60 plants of Helichrysum spp. were sampled on 18 June and 18 July 2020, at sites A and B, respectively. For each site, 30 plants were carefully selected randomly within the population to preserve the intra-site variability of the species. Then, for each plant, a portion of bark, vegetative shoot and full-blooming inflorescence was collected, following the protocol described by Squillace [27]. The collected samples were finally transported in refrigerated containers at 4 • C and then placed in the laboratory at −20 • C until terpene analysis. Moreover, for each site, 15 plants from each population were sampled to obtain exsiccata for morphological analysis.

Morphological Analysis
In this study, three variables were further analyzed compared to the 24 variables currently applied for the morphological description of the Helichrysum (Table 2). Specifically, the glandular indumentum of the leaf abaxial side (ID05); the axillary fascicles of the floral stem (ID21); and the ratio between synflorescence length and the number of capitula per synflorescence (ID27) were evaluated. In addition, for ID05, ID14, ID17, and ID20, a quantitative approach rather than only qualitative information was reported. For ID05 and ID20, the glandular indumentum of the proximal part of the leaf's abaxial side was also investigated.
Morphological measurements were performed using digital images on exsiccata at different resolution (i.e., 15×; 50×; 240×). All images were obtained using a DinoLite digital microscope (mod. AM4113ZT) and processed with the DinoCapture software (ver. 2.0) ( Table 2). For all characteristics, where possible, the mean of three to six measurements per specimen was used.

Micromorphological Analysis
Phytochemical analysis was carried out on cortex, vegetative shoot and full-blooming inflorescence by extraction through sonication and maceration in heptane.

Plant Materials
A total of 60 plants of Helichrysum spp. were sampled on 18 June and 18 July 2020, at sites A and B, respectively. For each site, 30 plants were carefully selected randomly within the population to preserve the intra-site variability of the species. Then, for each plant, a portion of bark, vegetative shoot and full-blooming inflorescence was collected, following the protocol described by Squillace [27]. The collected samples were finally transported in refrigerated containers at 4°C and then placed in the laboratory at −20°C until terpene analysis. Moreover, for each site, 15 plants from each population were sampled to obtain exsiccata for morphological analysis.

Morphological Analysis
In this study, three variables were further analyzed compared to the 24 variables currently applied for the morphological description of the Helichrysum (Table 2). Specifically, the glandular indumentum of the leaf abaxial side (ID05); the axillary fascicles of the floral

Terpene Analysis
Terpene analyses were performed three weeks from the sample collection of bark, vegetative shoot, and full-blooming inflorescence. The analysis was carried out by sonicating and macerating the samples in heptane. The samples were weighed in 1.5 mL vials (i.e., 0.01 g of bark, 0.02 g of the central part of the vegetative shoot and 0.02 g of full-blooming capitula) and extracted with 1 mL of heptane. Then, samples were vortex mixed and incubated in an ultrasonic bath (BandelinSonorex Super RK 102H) for 15 min three times and successively incubated at 30 • C with a rotary shaker at 10 revolutions per minute for 24 h. The extracted samples were centrifuged at 3000 rpm for 15 min. Finally, 1 µL of the supernatant was injected into GC-MS (Agilent model 7820A) equipped with a single quadrupole mass detector (model 5977E). The adapted analytical conditions were injector temperature 260 • C in spitless mode; 50 m HP-Innowax column, having 0.2 mm i.d. and 0.4 µm absorbent film thickness; helium carrier gas at a flow rate of 1.2 mL/minute in constant flow mode. The mass detector was set in scan mode in the m/z range 29-350, at a rate of 3 scans per second. GC-MS chromatograms were processed with "Agilent Mass Hunter Quantitative Analysis" software through deconvolution and dynamic background subtraction. Identification of compounds was performed by comparing their mass spectra with those reported in the National Institute of Standards and Technology (NIST 11, Gaithersburg, MD, USA) mass spectra library. "Tentative" identification was based on Kovats retention indices (KI) and identification was confirmed by comparison of KI and mass spectra with those of available standards. The relative amount (percentages) of each monoterpene was expressed as a percentage of total monoterpenes (monoterpene profiles), while the relative amount (percentages) of each sesquiterpene was expressed as a percentage of total monoterpenes (terpene profiles).

Statistical Analysis
Analysis of variance (ANOVA) was carried out both on morphometric and terpene data. The analysis was performed using R Studio v. 1.4 [67] and SYSTAT v 13.4 (Systat Software, Inc., San Jose, CA, USA, 2021). The distribution of qualitative morphological characters was analyzed by the chi-square test, whilst normality and homoscedasticity of quantitative variables were analyzed with Shapiro-Wilk and Levene test. Variables that did not fulfil ANOVA assumptions, even after transformation, were analyzed by Kruskal-Wallis's test or Welch's test.
Principal component analysis (PCA) was also carried out using R Studio with Fac-torMineR and Factorextra packages [68,69] for both morphological and terpene data. The correlation between significant ANOVA morphological traits and terpene compounds was provided by coefficients of Spearman and Pearson, respectively. The highly correlated variables (r > |0.95|) were excluded from analyses.

Conclusions
The primary objective of this work was to discriminate between the two wild populations of Helichrysum spp. utilizing morphological and phytochemical approaches. Morphological analysis revealed that leaf width and margin, the density of the glandular structure in the abaxial leaves indumentum and the size of cypselae were the main traits discriminating between the two species. Micromorphological analysis indicated the waxy microstructures and the density of the duplex-hair on the cypsela as the main characteristics discriminating between H. litoreum to H. italicum.
Terpene analysis revealed that, among all monoterpenes, the high content of nerylacetate could make it the most suitable compound to identify H. italicum, while high contents of α-pinene characterize H. litoreum. Among sesquiterpenes, the presence of a high relative percentage of α-curcumene, isoitalicene and italicene could be the most stable and discriminating compounds for H. italicum, while the high relative content of β-caryophyllene could be a possible marker for H. litoreum. Finally, bark tissue revealed its suitability in chemotaxonomic analysis, since the terpene profile of bark tissue provided more stable compounds that could be used as biochemical markers.
However, although our analysis reported numerous pieces of evidence, future studies are needed. First, an in-depth genetic analysis of Helichrysum populations would help to define the species distribution in the Tuscan Archipelago territory. In addition, to further refine the chemotaxonomic analysis, our future studies will analyze terpene profiles of seedlings of vegetatively propagated clones from different Italian populations of H. litoreum and H. italicum grown in an experimental plantation under the same environmental conditions. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/plants11131719/s1, Figure S1: Trichomes morphotypes. Figure S2: Correlation coefficient morphological data. Figure S3: Correlation coefficient: terpene flower data. Figure S4: Correlation coefficient: terpene leaves data. Figure S5: Correlation coefficient: terpene bark data. Table S1: Contribution of morphological characters to the principal components. Table S2: Phytochemical analysis of flower, leaves and bark tissue. Table S3: Contribution of flower phytochemical compounds to the principal components. Table S4: Contribution of leaves phytochemical compounds to the principal components. Table S5: Contribution of bark phytochemical compounds to the principal components.